Tumor cells escape recognition by the immune system by multiple mechanisms that include activation of inhibitory immune checkpoint pathways. Therapeutic inhibition of these immune checkpoints has demonstrated striking efficacy in a number of cancers and is a promising new approach to cancer treatment in combination with other anti-cancer regimens. Antibodies against the PD-1 pathway have in particular revolutionized the treatment of patients with advanced melanoma and produce tumor responses in >40% of patients. Combinations of anti-PD-1 with anti-CTLA-4 produces tumor responses in approximately 60% of patients. However, immune checkpoint blockade frequently causes inflammatory and immune-related Adverse Events (irAEs) due to the disruption of self-tolerance protection of normal tissues. These irAEs can be severe, lead to discontinuation of immune checkpoint inhibitor therapy and can require immunosuppressive treatment. Any tissue can be injured with the most frequent occurrences in the skin, gastro-intestinal tract, endocrine glands, liver, and lungs. Combined anti-PD-1 and CTLA-4 has significantly higher toxicity than monotherapy and requires more frequent and aggressive management. Although treatment with steroids and other immune modulators can reverse these irAEs, immunosuppression may compromise the anti-tumor activity of the checkpoint blockade. Thus, there is an unmet need for availability of clinically validated, non-invasive biomarkers for real time monitoring and prediction of on- or post-treatment irAEs and therapeutic efficacy to allow for proper management of cancer patients exposed to immune checkpoint inhibitors. Here we propose to monitor anti-tumor efficacy (Aim 1) as well as organ-specific irAEs (Aim 2) using cell-free DNA analysis from serial blood samples obtained before and at regular intervals during and after treatment. Under Aim 1 we propose to monitor changes in circulating cell-free mutant tumor DNA (ctDNA) patterns as a readout of anti- tumor treatment efficacy. Under Aim 2 we propose to assess autoimmune organ damage by monitoring changes in the abundance of circulating cell-free, tissue-specific methylated DNA (cmeDNA). We are currently leading a national cooperative group trial (EA6134) that involves combination anti-CTLA-4 and anti-PD-1 treatment of patients with BRAF mutant melanoma. Serially collected blood samples from patients on this trial will be analyzed as they respond to treatment, develop irAEs, require immunosuppressive therapy or discontinuation of treatment. We will compare the ctDNA and cmeDNA biomarker readouts with clinical observations of efficacy and adverse events in the trial to establish their utility.